Detection Of Salmonellae In Foods Research Articles

Rapid Detection of Salmonellae by Means of a New Impedance-Splitting Method

An Impedance-Splitting method is proposed for the rapid detection of salmonellae in foods. The measuring System, BacTrac™ 4100, permits the registration of changes, caused by bacterial metabolism, not only of the impedance of the culture medium but also of changes in the ionic layers at the measuring electrodes, which has advantages in case of high salt concentrations. These changes are expressed as percentage decreases of the initial values, M-value and E-value, respectively. Food samples were pre-enriched 14 to 16 h at 37°C in peptone water by addition of mannitol, which facilitated the detection of salmonellae on selective culture media. Following this, 0.1 mi of the preenrichment culture was transferred to 9.9 ml of Impedance-Splitting Salmonellae (ISS) medium which consisted of magnesium chloride (hydrated), malachite green oxalate, novobiocin, phosphate buffer, mannitol, peptone and yeast extract. Despite the high magnesium chloride concentration in this medium, salmonellae produced changes of the E-value up to 100%, while the changes in M-values were limited to a few percent. The impedance changes were automatically recorded during incubation in the measuring system for up to 22 h at 40°C, and the time required to exceed a threshold value of 15% (E reaction time) was evaluated. Comparative testing of the ISS method with standard cultural analysis of 250 unknown food samples showed high sensitivity and selectivity in detecting salmonellae. From all of the 122 Salmonella-positive samples, the largest number (119) was obtained by the ISS method, as compared to that obtained by conventional testing with the selenite-cystine (106), Rappaport Vassiliadis soya (95), Rappaport Vassiliadis (92) and tetrathionate brilliant green medium (64). Six samples were false positive by Enterobacter cloaceae. One strain each of Salmonella enteritidis PT8 and Salmonella panama were not recorded. The ISS method is very suitable as a screening test, all the more since a negative investigation result will be obtained within 38 h. In view of the practicability, this method is superior to the enzyme-immunological and molecular-biological procedures.

Comparative Study of Colorimetric DNA Hybridization Method and Conventional Culture Procedure for Detection of Salmonella in Foods

A second generation nucleic acid hybridization assay has been developed and evaluated against the conventional culture method for detection of salmonellae in foods. The assay involves a liquid hybridization with Salmonella-specific oligonucleotide probes, capture of probe:target hybrids onto a solid support (plastic dipstick), and a colorimetric end point detection. The assay can be completed in 2.5 h, following approximately 44 h of culture enrichment. One thousand samples representing 20 food types were analyzed in parallel by both methods. Samples included uninoculated test product, and product inoculated with Salmonella at 2 levels. Eighteen Salmonella serotypes were used as inocula. The data demonstrate that the colorimetric hybridization method and the conventional culture method are equivalent in their ability to detect Salmonella contamination of foods.

Immunodiffusion Screening Method for Detection of Motile Salmonella in Foods: Collaborative Study

A collaborative study was performed to validate the performance of the 1-2 TEST for detection of motile salmonellae in foods. Detection is based on observation of an immobilized band of cells. Twenty-three laboratories participated in the study. The 1-2 TEST (immunodiffusion test) was compared with the standard culture procedure (BAM/AOAC; FDA Bacteriological Analytical Manual) for detection of Salmonella in 6 food types: ground black pepper, soy flour, dried whole egg, milk chocolate, nonfat dry milk, and raw deboned turkey. Uninoculated and inoculated samples were included in each food group analyzed. After the tests on the 6 foods were completed, analysis of the data for turkey and soy flour showed that certain collaborators obtained data inconsistent with the data from the majority of collaborators. No specific method deviations to account for the inconsistencies were reported by those collaborators, so the collaborative testing of these 2 foods was repeated. Analysis of data for pepper, chocolate, nonfat dry milk, dried whole egg, and the second set of soy flour and turkey indicated 96.1% agreement between the BAM/AOAC and immunodiffusion test methods. The false negative rates for the immunodiffusion test and BAM/AOAC methods were 3.6 and 1.7%, respectively. There was no significant difference in the productivity of the immunodiffusion test and BAM/AOAC method at the 5% level for any of the 6 foods. The immunodiffusion screening method has been approved official first action for detection of motile Salmonella in foods.

Direct immunoassay for detection of salmonellae in foods and feeds.

A direct enzyme immunoassay (EIA) with polyclonal antibodies was developed for detecting salmonellae in foods and feeds. Salmonella cells were attached firmly to the wells of polystyrene microtitration plates with a capture-antibody technique. Spicer-Edwards anti-H immunoglobulin G was bound to protein A-beta-D-galactosidase to serve as the signal; 4-methylumbelliferyl-beta-D-galactoside was used as the substrate. The sensitivity threshold was 10(7) cells per ml. Direct EIA, indirect EIA, and pure-culture techniques were compared by using 48 samples of naturally contaminated foods and feeds. The direct EIA was more sensitive than the indirect EIA or pure-culture technique. Food samples were analyzed within 3 working days, and 32 samples were tested simultaneously in a single 96-well microtitration plate. False-positive or false-negative results did not pose a problem. This direct EIA is sensitive, rapid, and amenable to automation.

An enzyme immunoassay for the detection of all Salmonella using a combination of a myeloma protein and a hybridoma antibody

An enzyme immunoassay was developed for the detection of salmonellae in foods and stool samples. The test is sensitive and specific, showing virtually no cross-reactivity to other enteric organisms. The sensitivity is such that <10 6 Salmonella organisms can be detected, and this is not influenced even by a 3 log 10 overgrowth of other bacteria. This assay can be performed routinely in any laboratory, and can be automated as much as desired. It can be performed from growth in liquid or on solid media. A myeloma protein (M467) and a hybridoma antibody (6H4) were attached to a polycarbonate coated metal bead. The beads were then incubated in a heat extract of the organisms for 20 min. After thorough washing by a magnetic transfer device, the beads were then incubated in a mixture of the 2 antibodies which were labeled with horseradish peroxidase. After another thorough washing and incubation in substrate, the reactions were either read by the naked eye or by spectrophotometry. The entire process following the culture takes less than 2 h.

Effective Enrichment-Plating Conditions for Detection of Salmonella in Foods

The performance of tetrathionate brilliant green (TBG) and selenite cystine (SC) enrichments, and bismuth sulfite (BSA) and brilliant green sulfa (BGS) plating media was assessed on the basis of data on 2085 Salmonella-contaminated low and high moisture foods that were collected during a 6-year (1977 to 1983) study involving 22 laboratories. None of the eight enrichment- plating combinations considered identified all positive samples. TBG was markedly more sensitive than SC for detection of salmonellae in high moisture foods, where enrichment in TBG and plating on both BSA and BGS identified 92% of contaminated samples. Plating of SC enrichment cultures on the two agar media was substantially less productive (63%). With low moisture foods, TBG and SC rates of isolation varied by less than 10% under homologous conditions. Plating media did not affect method sensitivity. Identification of numerous contaminated samples by TBG or SC enrichment alone underlines the value of using multiple enrichment and plating media in standard methods.

Salmonella detection in foods and feeds in 27 hours by an enzyme immunoassay

A heterogeneous enzyme immunoassay (EIA), which could be completed within 27 h, was developed for the detection of salmonellae in foods. Samples were subjected to the usual non-selective enrichment for 16–18 at 35°C and to a short (6 h) post-enrichment in a moderately selective broth. The EIA was carried out on polystyrene microtitration plates. A pooled polyvalent Salmonella flagellar antiserum and a protein A-alkaline phosphatase conjugate were used. The sensitivity of the enzyme immunoassay compared favorably with that of the conventional cultural technique for detection of Salmonella in 40 naturally contaminated food and feed samples. No sample was positive only by the cultural technique; samples positive only by the enzyme immunoassay were observed for feeds. Some specimens yielded high background values indicating possible interference from food proteins.

An Improved Enzyme Immunoassay for the Detection of Salmonella

ABSTRACTAn enzyme immunoassay (EIA) was developed for the detection of salmonellae in foods or other samples. This assay detected Salmonella contamination within two working days after initiation, and the EIA required &lt;2 hr to perform. The test was sensitive and specific, showing virtually no cross reactivity to other organisms. No selective enrichment was found to be necessary, as the presence of other organisms, even in much greater numbers, had no effect on the reaction. The sensitivity is such that &lt;106Salmonella/mL were detected, and the food samples studied showed no effect on the background values and did not cause false readings.

RAPID DETECTION OF SALMONELLAE IN FOODS BY MEMBRANE FILTER‐DISC IMMUNOIMMOBILIZATION TECHNIQUE

ABSTRACTA membrane filter‐disc immunoimmobilization technique was developed for rapid detection of salmonellae in foods. The method involved concentrating bacteria from the selective enrichment culture of a food sample by membrane filtration. The membrane filter, with the entrapped bacteria, was inverted and placed on the surface of a semi‐solid selective medium contained in a 100 × 15 mm plastic Petri plate. A paper disc impregnated with Salmonella polyvalent flagellar antiserum was placed on the surface of the semi‐solid agar approximately 2.5 cm from the nearest edge of the membrane filter. The plate was incubated at 37° C under high humidity. Motile salmonellae, if present in the sample, grew and migrated in the semi‐solid medium. When the moving front of motile salmonellae came into contact with the diffusing flagellar antiserum, an antigen‐antibody reaction occurred resulting in the immobilization of salmonellae. The formation of a line of immobilization indicated the presence of salmonellae in the sample. A semi‐solid medium containing dulcitol, proteose peptone, brilliant green, and novobiochin as the major functional components was found to be more efficient than semi‐solid modifications of Salmonella‐Shigella agar and Hektoen enteric agar for the detection of salmonellae in foods by the membrane filter‐disc immunoimmobilization procedure. The new method, when applied to the detection of salmonellae in raw meats and poultry, was found to give good correlation with the conventional cultural method.

Control of nonspecific staining in the fluorescent antibody technique for the detection of salmonellae in foods

A fluorescent antibody conjugate, prepared from the IgG (immunoglobulin G) fraction of Salmonella polyvalent flagellar antiserum, gave better specific staining intensities and significantly lower nonspecific staining than did conjugates prepared from globulin fractions of ammonium sulfate-fractionated Salmonella polyvalent antisera. IgG was purified by affinity chromatography against protein A, a normal cell wall component of Staphylococcus aureus. Affinity chromatography yielded high-purity IgG in a one-step purification procedure. The conjugate prepared from affinity-purified IgG was compared with commercially available fluorescent antibody conjugates for the detection of salmoneallae in retail samplings of meats and poultry and gave better correlations with the cultural method than did the commercial conjugates.

Evaluation of an automated fluorescent antibody procedure for detection of Salmonella in foods and feeds

A prototype automated system using fluorescent antibody (FA) was evaluated for rapid detection of salmonellae in foods. Samples were enriched in selenite cystine and tetrathionate broths. After incubation, both were transferred into fresh selenite cystine for a 4-h "post-enrichment" to dilute possible background fluorescence from product. These cultures were then analyzed automatically, and results were compared with those obtained by the methods of the Association of Official Analytical Chemists (AOAC). Initially, 167 samples of milk powder, dried yeast, and imported frog legs were examined. The AOAC and automated FA methods correlated well with all samples but frog legs. Difficulty with the latter was caused by procedural and mechanical problems coupled with high numbers of competing microorganisms in post-enrichment cultures. Modification of procedure and partial redesign of equipment corrected these difficulties, and excellent correlation was obtained with another 116 frog leg samples. All 89 AOAC-confirmed positives were also detected by the automated FA method, and there were only 4% false FA positives. The system shows potential for screening products for salmonellae; however, all positives should be confirmed by manual biochemical and serological methods.

Evaluation of Hektoen Enteric Agar for the Detection of Salmonella in Foods and Feeds

Abstract Over the past 2 years in our laboratory, Hektoen enteric (HE) agar was evaluated as a medium for the detection of salmonellae in foods and feeds. HE agar was used in conjunction with brilliant green, bismuth sulfite, and Salmonella-Shigella agars, according to AOAC methods. More than 2000 samples representing over 40 different products were tested and the cultural results on all media were compared. HE agar was more efficient than the other media for recovery of salmonellae in contaminated samples and showed a lower number of false positives. HE agar is a highly selective medium for Salmonella species and we recommend that it be evaluated for incorporation into the AOAC methods.

Rapid, direct fluorescent-antibody method for the detection of salmonellae in food and feeds.

An improved immunofluorescent-antibody (FA) method for the detection of salmonellae in foods and feeds was developed. This FA method combines a rapid cultural phase and a serological phase that allow for propagation of salmonellae in a minimum time, employing the industrial 8-hr work day as a guide. Two hundred fifty naturally contaminated human food and animal feed samples, representing 647 trials, were tested by the FA method. A total of 18 different food and feed samples was used. The method used by the Association of Official Analytical Chemists (AOAC) for the detection of salmonellae was the control method. The percent agreement when comparing the FA slide method to the AOAC method ranged from 87.1 to 95.3%, depending upon the conjugated antisera used in comparative studies.

Rapid, Direct Fluorescent-Antibody Method for the Detection of Salmonellae in Food and Feeds

An improved immunofluorescent-antibody (FA) method for the detection of salmonellae in foods and feeds was developed. This FA method combines a rapid cultural phase and a serological phase that allow for propagation of salmonellae in a minimum time, employing the industrial 8-hr work day as a guide. Two hundred fifty naturally contaminated human food and animal feed samples, representing 647 trials, were tested by the FA method. A total of 18 different food and feed samples was used. The method used by the Association of Official Analytical Chemists (AOAC) for the detection of salmonellae was the control method. The percent agreement when comparing the FA slide method to the AOAC method ranged from 87.1 to 95.3%, depending upon the conjugated antisera used in comparative studies.

Comparison of Two Methods for the Isolation of Salmonellae From Imported Foods

Two methods for the detection of salmonellae in foods were compared in 179 imported meat and egg samples. The number of positive samples and replications, and the number of strains and kinds of serotypes were statistically comparable by both the direct enrichment method of the Food Hygiene Laboratory in England, and the pre-enrichment method devised for processed foods in the United States. Boneless frozen beef, veal, and horsemeat imported from five countries for consumption in England were found to have salmonellae present in 48 of 116 (41%) samples. Dried egg products imported from three countries were observed to have salmonellae in 10 of 63 (16%) samples. The high incidence of salmonellae isolated from imported foods illustrated the existence of an international health hazard resulting from the continuous introduction of exogenous strains of pathogenic microorganisms on a large scale.

COMPARISON OF TWO METHODS FOR THE ISOLATION OF SALMONELLAE FROM IMPORTED FOODS.

Two methods for the detection of salmonellae in foods were compared in 179 imported meat and egg samples. The number of positive samples and replications, and the number of strains and kinds of serotypes were statistically comparable by both the direct enrichment method of the Food Hygiene Laboratory in England, and the pre-enrichment method devised for processed foods in the United States. Boneless frozen beef, veal, and horsemeat imported from five countries for consumption in England were found to have salmonellae present in 48 of 116 (41%) samples. Dried egg products imported from three countries were observed to have salmonellae in 10 of 63 (16%) samples. The high incidence of salmonellae isolated from imported foods illustrated the existence of an international health hazard resulting from the continuous introduction of exogenous strains of pathogenic microorganisms on a large scale.